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Dennis E Discher

Publications and source records attributed to Dennis E Discher.

10 recordsLinked to original sources
PubMed2003

Protein 4.2 is critical to CD47-membrane skeleton attachment in human red cells.

The reduction in expression of the integral membrane protein CD47 in human red blood cells (RBCs) deficient in protein 4.2 suggests that protein 4.2 may mediate a linkage of CD47 to the membrane skeleton. We compared the fractions of membrane skeleton-attached CD47, Rh-associated glycoprotein (RhAG), Rh, and band 3 in normal and protein 4.2-deficient cells using fluorescence-imaged microdeformation. We found that CD47 attachment decreases from 55% in normal cells to 25% to 35% in 4.2-deficient cells. RhAG, which has been shown to have no significant variation in expression among the cells studied, shows a significant decrease in membrane skeleton attachment in 4.2-deficient cells from 60% to 40%. Both Rh and band 3, which have also been shown to have no change in expression, show a smaller decrease from 75% attached in normal RBCs to 55% attached in 4.2-deficient cells. In normal cells, Rh phenotype influences CD47 expression but not the level of membrane skeleton attachment of CD47. In contrast, the results indicate that protein 4.2 strongly influences CD47 levels as well as the extent of membrane skeleton attachment in the RBC, whereas protein 4.2 affects membrane skeletal attachment of RhAG, Rh, and band 3 to a lesser extent.

Actins↗
PubMed2003

Polymer vesicles in vivo: correlations with PEG molecular weight.

PEG-modified lipid vesicles have already shown considerable utility in delaying vesicle clearance from the circulation. They are, however, limited in their ability to stably integrate high molar ratios of PEG-lipid due to the high curvature and micellar preference of the very large hydrophilic PEG chain. Polymersomes, by contrast, are vesicles composed entirely of PEG-based block copolymer amphiphiles that are not only more proportionately designed, but also have already been shown to considerably broaden the range of vesicle properties (e.g. stability). Here, polymersomes composed of varying length copolymer chains were injected into rats and found to have in vivo circulation times, tau(1/2), up to about two-fold longer than PEGylated, or Stealth, liposomes. The dependence of tau(1/2) on PEG molecular weight is nonetheless limited by uptake into the liver and spleen-as with liposomes. In vitro incubations of polymersomes in plasma indicate gradual opsonization through plasma protein adsorption, such that, when vesicles are held in an optical trap and presented to a phagocyte, rapid engulfment occurs only after incubation times of similar magnitude to tau(1/2). The stealthiness introduced to liposomes through PEGylation is thus extended here with completely synthetic polymersomes.

Animals↗
PubMed2003

Networks with fourfold connectivity in two dimensions.

The elastic properties of planar, C4-symmetric networks under stress and at nonzero temperature are determined by simulation and mean field approximations. Attached at fourfold coordinated junction vertices, the networks are self-avoiding in that their elements (or bonds) may not intersect each other. Two different models are considered for the potential energy of the elements: either Hooke's law springs or flexible tethers (square well potential). For certain ranges of stress and temperature, the properties of the networks are captured by one of several models: at large tensions, the networks behave like a uniform system of square plaquettes, while at large compressions or high temperatures, they display many characteristics of an ideal gas. Under less severe conditions, mean field models with more general shapes (parallelograms) reproduce many essential features of both networks. Lastly, the spring network expands without limit at a two-dimensional tension equal to the force constant of the spring; however, it does not appear to collapse under compression, except at zero temperature.

Animals↗
PubMed2003

Adhesively-tensed cell membranes: lysis kinetics and atomic force microscopy probing.

Membrane tension underlies a range of cell physiological processes. Strong adhesion of the simple red cell is used as a simple model of a spread cell with a finite membrane tension-a state which proves useful for studies of both membrane rupture kinetics and atomic force microscopy (AFM) probing of native structure. In agreement with theories of strong adhesion, the cell takes the form of a spherical cap on a substrate densely coated with poly-L-lysine. The spreading-induced tension, sigma, in the membrane is approximately 1 mN/m, which leads to rupture over many minutes; and sigma is estimated from comparable rupture times in separate micropipette aspiration experiments. Under the sharpened tip of an AFM probe, nano-Newton impingement forces (10-30 nN) are needed to penetrate the tensed erythrocyte membrane, and these forces increase exponentially with tip velocity ( approximately nm/ms). We use the results to clarify how tapping-mode AFM imaging works at high enough tip velocities to avoid rupturing the membrane while progressively compressing it to a approximately 20-nm steric core of lipid and protein. We also demonstrate novel, reproducible AFM imaging of tension-supported membranes in physiological buffer, and we describe a stable, distended network consistent with the spectrin cytoskeleton. Additionally, slow retraction of the AFM tip from the tensed membrane yields tether-extended, multipeak sawtooth patterns of average force approximately 200 pN. In sum we show how adhesive tensioning of the red cell can be used to gain novel insights into native membrane dynamics and structure.

Cell Count↗
PubMed2003

Pathway shifts and thermal softening in temperature-coupled forced unfolding of spectrin domains.

Pathways of unfolding a protein depend in principle on the perturbation-whether it is temperature, denaturant, or even forced extension. Widely-shared, helical-bundle spectrin repeats are known to melt at temperatures as low as 40-45 degrees C and are also known to unfold via multiple pathways as single molecules in atomic force microscopy. Given the varied roles of spectrin family proteins in cell deformability, we sought to determine the coupled effects of temperature on forced unfolding. Bimodal distributions of unfolding intervals are seen at all temperatures for the four-repeat beta(1-4) spectrin-an alpha-actinin homolog. The major unfolding length corresponds to unfolding of a single repeat, and a minor peak at twice the length corresponds to tandem repeats. Increasing temperature shows fewer tandem events but has no effect on unfolding intervals. As T approaches T(m), however, mean unfolding forces in atomic force microscopy also decrease; and circular dichroism studies demonstrate a nearly proportional decrease of helical content in solution. The results imply a thermal softening of a helical linker between repeats which otherwise propagates a helix-to-coil transition to adjacent repeats. In sum, structural changes with temperature correlate with both single-molecule unfolding forces and shifts in unfolding pathways.

Circular Dichroism↗
PubMed2003

Cooperativity in forced unfolding of tandem spectrin repeats.

Force-driven conformational changes provide a broad basis for protein extensibility, and multidomain proteins broaden the possibilities further by allowing for a multiplicity of forcibly extended states. Red cell spectrin is prototypical in being an extensible, multidomain protein widely recognized for its contribution to erythrocyte flexibility. Atomic force microscopy has already shown that single repeats of various spectrin family proteins can be forced to unfold reversibly under extension. Recent structural data indicates, however, that the linker between triple-helical spectrin repeats is often a contiguous helix, thus raising questions as to what the linker contributes and what defines a domain mechanically. We have examined the extensible unfolding of red cell spectrins as monomeric constructs of just two, three, or four repeats from the actin-binding ends of both alpha- and beta-chains, i.e., alpha(18-21) and beta(1-4) or their subfragments. In addition to single repeat unfolding evident in sawtooth patterns peaked at relatively low forces (<50 pN at 1 nm/ms extension rates), tandem repeat unfolding is also demonstrated in ensemble-scale analyses of thousands of atomic force microscopy contacts. Evidence for extending two chains and loops is provided by force versus length scatterplots which also indicate that tandem repeat unfolding occurs at a significant frequency relative to single repeat unfolding. Cooperativity in forced unfolding of spectrin is also clearly demonstrated by a common force scale for the unfolding of both single and tandem repeats.

Elasticity↗
PubMed2002

Domain unfolding in neurofilament sidearms: effects of phosphorylation and ATP.

Lateral projections of neurofilaments (NF) called sidearms (SA) affect axon stability and caliber. SA phosphorylation is thought to modulate inter-NF distance and interactions between NF and other subcellular organelles. SA were probed by atomic force microscopy (AFM) and dynamic light scattering (DLS) as a function of phosphorylation and ATP content. DLS shows SA are larger when phosphorylated, and AFM shows four unfoldable domains in SA regardless of phosphorylation state or the presence of ATP. However, the native phosphorylated SA requires three-fold higher force to unfold by AFM than dephosphorylated SA, suggesting a less pliant as well as larger structure when phosphorylated.

Adenosine Triphosphate↗
PubMed2002

Fractional attachment of CD47 (IAP) to the erythrocyte cytoskeleton and visual colocalization with Rh protein complexes.

Interactions of CD47 and RhAG and the Rh proteins are visualized between one another and with the cytoskeleton of intact erythrocytes. In a first study, CD47 is labeled with a phycoerythrin (PhE)- tagged antibody, which generates discrete spots that reflect induced clusters of CD47. Rh and RhAG colocalize with each other and to these induced clusters, whereas Band 3 and glycophorin C remain more homogeneously dispersed on the cell periphery. In a second study, red cells are aspirated into a micropipette, and immunofluorescent maps of the surface gradients that develop for CD47 and RhAG determine cytoskeletal connectivity. CD47 and RhAG gradients on normal red cells prove to be nearly identical and also appear intermediate to those found for the fluid bilayer and network-linked glycophorin C. Similar gradients are obtained for CD47 on Rh(null) cells, suggesting that linkage of CD47 to the spectrin-actin skeleton is independent of Rh or RhAG and is not affected by CD47's reduced surface expression on these cells. The results show that CD47 colocalizes with Rh and RhAG but is fractionally attached to the red cell membrane skeleton independent of these and other major integral membrane proteins involved in cytoskeletal attachment. The results imply a homogeneous base distribution of CD47, restrained by cytoskeleton linkages, plus a smaller fraction of CD47, which is able to diffuse in the membrane.

Anion Exchange Protein 1, Erythrocyte↗
PubMed2002

Polymer vesicles.

Vesicles are microscopic sacs that enclose a volume with a molecularly thin membrane. The membranes are generally self-directed assemblies of amphiphilic molecules with a dual hydrophilic-hydrophobic character. Biological amphiphiles form vesicles central to cell function and are principally lipids of molecular weight less than 1 kilodalton. Block copolymers that mimic lipid amphiphilicity can also self-assemble into vesicles in dilute solution, but polymer molecular weights can be orders of magnitude greater than those of lipids. Structural features of vesicles, as well as properties including stability, fluidity, and intermembrane dynamics, are greatly influenced by characteristics of the polymers. Future applications of polymer vesicles will rely on exploiting unique property-performance relations, but results to date already underscore the fact that biologically derived vesicles are but a small subset of what is physically and chemically possible.

Animals↗